Transcriptomic profiling of adaptive responses to ocean acidification.

Some populations of marine organisms appear to have inherent tolerance or the capacity for acclimation to stressful environmental conditions, including those associated with climate change. Sydney rock oysters from the B2 breeding line exhibit resilience to ocean acidification (OA) at the physiological level. To understand the molecular basis of this physiological resilience, we analysed the gill transcriptome of B2 oysters that had been exposed to near-future projected ocean pH over two consecutive generations. Our results suggest that the distinctive performance of B2 oysters in the face of OA is mediated by the selective expression of genes involved in multiple cellular processes. Subsequent high-throughput qPCR revealed that some of these transcriptional changes are exclusive to B2 oysters and so may be associated with their resilience to OA. The intracellular processes mediated by the differentially abundant genes primarily involve control of the cell cycle and maintenance of cellular homeostasis. These changes may enable B2 oysters to prevent apoptosis resulting from oxidative damage or to alleviate the effects of apoptosis through regulation of the cell cycle. Comparative analysis of the OA conditioning effects across sequential generations supported the contention that B2 and wild-type oysters have different trajectories of changing gene expression and responding to OA. Our findings reveal the broad set of molecular processes underlying transgenerational conditioning and potential resilience to OA in a marine calcifier. Identifying the mechanisms of stress resilience can uncover the intracellular basis for these organisms to survive and thrive in a rapidly changing ocean.

Double stranded RNA is processed differently in two oyster species.

Ostreid herpes virus causes serious disease in the Pacific oyster (Crassostrea gigas), but not in the Sydney Rock Oyster (Saccostrea glomerata). To investigate differences in disease progression, we injected oysters with double stranded RNA (dsRNA). dsRNA is known to mimic viral infection, and can evoke immune responses when Toll-like receptors detect the dsRNA, leading to the production of type 1 interferon and inflammation cytokines. The uptake and processing of dsRNA was tracked in gill and mantle tissue of Crassostrea gigas and Saccostrea glomerata after injection of fluorochrome labelled poly (I:C) dsRNA. The two species showed significant differences in tissue uptake and clearance, and differences in immune responses confirmed by real time PCR. These results showed that S. glomerata was more efficient in processing dsRNA than C. gigas, and that the gill tissue is an important site of dsRNA processing and response.

Contrasting impacts of ocean acidification and warming on the molecular responses of CO2-resilient oysters.

BACKGROUND: This study characterises the molecular processes altered by both elevated CO2 and increasing temperature in oysters. Differences in resilience of marine organisms against the environmental stressors associated with climate change will have significant implications for the sustainability of coastal ecosystems worldwide. Some evidence suggests that climate change resilience can differ between populations within a species. B2 oysters represent a unique genetic resource because of their capacity to better withstand the impacts of elevated CO2 at the physiological level, compared to non-selected oysters from the same species (Saccostrea glomerata). Here, we used proteomic and transcriptomic analysis of gill tissue to evaluate whether the differential response of B2 oysters to elevated CO2 also extends to increased temperature. RESULTS: Substantial and distinctive effects on protein concentrations and gene expression were evident among B2 oysters responding to elevated CO2 or elevated temperature. The combination of both stressors also altered oyster gill proteomes and gene expression. However, the impacts of elevated CO2 and temperature were not additive or synergistic, and may be antagonistic. CONCLUSIONS: The data suggest that the simultaneous exposure of CO2-resilient oysters to near-future projected ocean pH and temperature results in complex changes in molecular processes in order to prevent stress-induced cellular damage. The differential response of B2 oysters to the combined stressors also indicates that the addition of thermal stress may impair the resilience of these oysters to decreased pH. Overall, this study reveals the intracellular mechanisms that might enable marine calcifiers to endure the emergent, adverse seawater conditions resulting from climate change.

Shifting paradigms in restoration of the world's coral reefs.

Many ecosystems around the world are rapidly deteriorating due to both local and global pressures, and perhaps none so precipitously as coral reefs. Management of coral reefs through maintenance (e.g., marine-protected areas, catchment management to improve water quality), restoration, as well as global and national governmental agreements to reduce greenhouse gas emissions (e.g., the 2015 Paris Agreement) is critical for the persistence of coral reefs. Despite these initiatives, the health and abundance of corals reefs are rapidly declining and other solutions will soon be required. We have recently discussed options for using assisted evolution (i.e., selective breeding, assisted gene flow, conditioning or epigenetic programming, and the manipulation of the coral microbiome) as a means to enhance environmental stress tolerance of corals and the success of coral reef restoration efforts. The 2014-2016 global coral bleaching event has sharpened the focus on such interventionist approaches. We highlight the necessity for consideration of alternative (e.g., hybrid) ecosystem states, discuss traits of resilient corals and coral reef ecosystems, and propose a decision tree for incorporating assisted evolution into restoration initiatives to enhance climate resilience of coral reefs.

Primed for success: Oyster parents treated with poly(I:C) produce offspring with enhanced protection against Ostreid herpesvirus type I infection.

The Pacific oyster (Crassostrea gigas) is farmed globally. Ostreid herpesvirus (OsHV-1) causes severe mortalities of farmed C. gigas. Management of OsHV-1 has proven difficult. Oysters treated with poly(I:C) exhibit enhanced protection (EP) against OsHV-1. This chemical treatment is highly effective, but it is not feasible to treat every oyster on a farm. To circumvent this practical limitation, previous studies on arthropods have suggested that EP can be transferred from parents to their offspring (trans-generational EP, TGEP). This suggests that the treatment of relatively few parents could be used to produce large numbers of offspring with TGEP. Here, we investigated TGEP in oysters to test whether it might be used as a cost effective management tool to control OsHV-1. We found that offspring (D-veliger larvae) produced from poly(I:C)-treated parents had double the chance of surviving exposure to OsHV-1 compared to controls. Furthermore, the larvae of poly(I:C)-treated parents contained elevated levels of mRNA encoding a key transcription factor that regulates antiviral immunity (IRF2). Poly(I:C) treatment had no effect on the survival of oyster parents. Hence, the enhanced immunity of their offspring could not be explained by genetic selection, and instead may reflect epigenetic reprogramming or maternal provisioning.

Rapid transcriptional acclimation following transgenerational exposure of oysters to ocean acidification.

Marine organisms need to adapt in order to cope with the adverse effects of ocean acidification and warming. Transgenerational exposure to CO2 stress has been shown to enhance resilience to ocean acidification in offspring from a number of species. However, the molecular basis underlying such adaptive responses is currently unknown. Here, we compared the transcriptional profiles of two genetically distinct oyster breeding lines following transgenerational exposure to elevated CO2 in order to explore the molecular basis of acclimation or adaptation to ocean acidification in these organisms. The expression of key target genes associated with antioxidant defence, metabolism and the cytoskeleton was assessed in oysters exposed to elevated CO2 over three consecutive generations. This set of target genes was chosen specifically to test whether altered responsiveness of intracellular stress mechanisms contributes to the differential acclimation of oyster populations to climate stressors. Transgenerational exposure to elevated CO2 resulted in changes to both basal and inducible expression of those key target genes (e.g. ecSOD, catalase and peroxiredoxin 6), particularly in oysters derived from the disease-resistant, fast-growing B2 line. Exposure to CO2 stress over consecutive generations produced opposite and less evident effects on transcription in a second population that was derived from wild-type (nonselected) oysters. The analysis of key target genes revealed that the acute responses of oysters to CO2 stress appear to be affected by population-specific genetic and/or phenotypic traits and by the CO2 conditions to which their parents had been exposed. This supports the contention that the capacity for heritable change in response to ocean acidification varies between oyster breeding lines and is mediated by parental conditioning.

Two Oyster Species That Show Differential Susceptibility to Virus Infection Also Show Differential Proteomic Responses to Generic dsRNA.

Viral diseases are a significant cause of mortality and morbidity in oysters, resulting in significant economic losses. We investigated the proteomic responses of these two species of oysters to generic double-stranded RNAs (poly I:C and poly A:U). Analysis of proteomic data using isobaric tags for relative and absolute quantitaion (iTRAQ) indicated that there were significant differences in the proteomic responses of the two oyster species resulting from this treatment. Gene ontology analysis showed that several biological processes, cellular components, and molecular function were unique to the different data sets. For example, a number of proteins implicated in the TLR signaling pathway were associated with the Saccostrea glomerata data set but were absent in the Crassostra gigas data set. These results suggest that the differences in the proteomic responses to dsRNA may underpin the biological differences in viral susceptibility. Molecular targets previously shown to be expressed in C. gigas in response to OsHV1 infections were not present in our proteomic data sets, although they were present in the RNA extracted from the very same tissues. Taken together, our data indicate that there are substantial disparities between transcriptomic and proteomic responses to dsRNA challenge, and a comprehensive account of the oysters' biological responses to these treatments must take into account that disparity.

Proteomic analysis of hemolymph from poly(I:C)-stimulated Crassostrea gigas.

Synthetic double stranded RNA (Poly(I:C)) injection of Crassostrea gigas results in a systemic antiviral response involving many evolutionary conserved antiviral effectors (ISGs). Compared to mammals, the timing of C. gigas ISG expression to viral or poly(I:C) injection is delayed (>12 h p.i.). It could be interpreted that a cytokine is responsible for the systemic, but delayed expression of C. gigas ISGs. We therefore analysed the acellular fraction of C. gigas hemolymph by two-dimensional electrophoresis (2-DE) to identify hemolymph proteins induced by poly(I:C). Poly(I:C) injection increased the relative intensity of four protein spots. These protein spots were identified by tandem mass spectrometry (LC-MS/MS) as a small heat shock protein (sHSP), poly(I:C)-inducible protein 1 (PIP1) and two isoforms of C1q-domain containing protein (C1qDC). RT-qPCR analysis confirmed that the genes encoding these proteins are induced in hemocytes of C. gigas injected with poly(I:C) (p < 0.05). Proteomic data from this experiment corroborates previous microarray and whole transcriptome studies that have reported up-regulation of C1qDC and sHSP during mass mortality events among farmed oysters.

Persistence of Positive Carryover Effects in the Oyster, Saccostrea glomerata, following Transgenerational Exposure to Ocean Acidification.

Ocean acidification (OA) is predicted to have widespread implications for marine organisms, yet the capacity for species to acclimate or adapt over this century remains unknown. Recent transgenerational studies have shown that for some marine species, exposure of adults to OA can facilitate positive carryover effects to their larval and juvenile offspring that help them to survive in acidifying oceanic conditions. But whether these positive carryover effects can persist into adulthood or the next generation is unknown. Here we tested whether positive carryover effects found in larvae of the oyster, Saccostrea glomerata following transgenerational exposure to elevated CO2, could persist into adulthood and whether subsequent transgenerational exposure of adults to elevated CO2 would facilitate similar adaptive responses in the next generation of larvae and juveniles. Following our previous transgenerational exposure of parental adults and first generation (F1) larvae to ambient (385 µatm) and elevated (856 µatm) CO2, newly settled F1 juveniles were transferred to the field at ambient CO2 for 14 months, until they reached reproductive maturity. At this time, the F1 adults were returned to the laboratory and the previous transgenerational CO2 exposure was repeated to produce F2 offspring. We found that the capacity of adults to regulate extracellular pH at elevated CO2 was improved if they had a prior history of transgenerational exposure to elevated CO2. In addition, subsequent transgenerational exposure of these adults led to an increase in the resilience of their larval and juvenile offspring. Offspring with a history of transgenerational exposure to elevated CO2 had a lower percentage abnormality, faster development rate, faster shell growth and increased heart rate at elevated CO2 compared with F2 offspring with no prior history of exposure to elevated CO2. Our results suggest that positive carryover effects originating during parental and larval exposure will be important in mediating some of the impacts of OA for later life-history stages and generations.

Meta-analysis of studies using suppression subtractive hybridization and microarrays to investigate the effects of environmental stress on gene transcription in oysters.

Many microarray and suppression subtractive hybridization (SSH) studies have analyzed the effects of environmental stress on gene transcription in marine species. However, there have been no unifying analyses of these data to identify common stress response pathways. To address this shortfall, we conducted a meta-analysis of 14 studies that investigated the effects of different environmental stressors on gene expression in oysters. The stressors tested included chemical contamination, hypoxia and infection, as well as extremes of temperature, pH and turbidity. We found that the expression of over 400 genes in a range of oyster species changed significantly after exposure to environmental stress. A repeating pattern was evident in these transcriptional responses, regardless of the type of stress applied. Many of the genes that responded to environmental stress encoded proteins involved in translation and protein processing (including molecular chaperones), the mitochondrial electron transport chain, anti-oxidant activity and the cytoskeleton. In light of these findings, we put forward a consensus model of sub-cellular stress responses in oysters.

Dose-dependent effects of metals on gene expression in the sydney rock oyster, Saccostrea glomerata.

In the current study, we tested the effects of common environmental contaminants (the metals zinc and lead) on gene expression in Sydney rock oysters (Saccrostrea glomerata). Oysters were exposed to a range of metal concentrations under controlled laboratory conditions. The expression of 14 putative stress response genes was then measured using quantitative, real-time (q) PCR. The expression of all 14 genes was significantly affected (p < 0.05 vs. nonexposed controls) by at least one of the metals, and by at least one dose of metal. For 5 of the 14 target genes (actin, calmodulin, superoxide dismutase, topoisomerase I, and tubulin) the alteration of expression relative to controls was highest at intermediate (rather than high) doses of metals. Such responses may reflect adaptive (acclimation) reactions in gene expression at low to intermediate doses of contaminants, followed by a decline in expression resulting from exposure at higher doses. The data are discussed in terms of the intracellular pathways affected by metal contamination, and the relevance of such gene expression data to environmental biomonitoring.

Characterization of the highly variable immune response gene family, He185/333, in the sea urchin, Heliocidaris erythrogramma.

This study characterizes the highly variable He185/333 genes, transcripts and proteins in coelomocytes of the sea urchin, Heliocidaris erythrogramma. Originally discovered in the purple sea urchin, Strongylocentrotus purpuratus, the products of this gene family participate in the anti-pathogen defenses of the host animals. Full-length He185/333 genes and transcripts are identified. Complete open reading frames of He185/333 homologues are analyzed as to their element structure, single nucleotide polymorphisms, indels and sequence repeats and are subjected to diversification analyses. The sequence elements that compose He185/333 are different to those identified for Sp185/333. Differences between Sp185/333 and He185/333 genes are also evident in the complexity of the sequences of the introns. He185/333 proteins show a diverse range of molecular weights on Western blots. The observed sizes and pIs of the proteins differ from predicted values, suggesting post-translational modifications and oligomerization. Immunofluorescence microscopy shows that He185/333 proteins are mainly located on the surface of coelomocyte subpopulations. Our data demonstrate that He185/333 bears the same substantial characteristics as their S. purpuratus homologues. However, we also identify several unique characteristics of He185/333 (such as novel element patterns, sequence repeats, distribution of positively-selected codons and introns), suggesting species-specific adaptations. All sequences in this publication have been submitted to Genbank (accession numbers JQ780171-JQ780321) and are listed in table S1.

Next generation sequence analysis of the transcriptome of Sydney rock oysters (Saccostrea glomerata) exposed to a range of environmental stressors.

Sydney rock oysters (Saccostrea glomerata) were exposed to environmental stressors at contaminated field sites or in a controlled laboratory setting. RNA seq transcriptome data were generated for the gill and digestive gland using Roche's 454 pyrosequencing technology. 28,685 contigs were de novo assembled which encoded 11,671 different protein products. The data will act as a reference for future studies in ecology, immunology and environmental toxicology.

Infectious microbial diseases and host defense responses in Sydney rock oysters.

Aquaculture has long been seen as a sustainable solution to some of the world's growing food shortages. However, experience over the past 50 years indicates that infectious diseases caused by viruses, bacteria, and eukaryotes limit the productivity of aquaculture. In extreme cases, these types of infectious agents threaten the viability of entire aquaculture industries. This article describes the threats from infectious diseases in aquaculture and then focuses on one example (QX disease in Sydney rock oysters) as a case study. QX appears to be typical of many emerging diseases in aquaculture, particularly because environmental factors seem to play a crucial role in disease outbreaks. Evidence is presented that modulation of a generic subcellular stress response pathway in oysters is responsible for both resistance and susceptibility to infectious microbes. Understanding and being able to manipulate this pathway may be the key to sustainable aquaculture.

No more non-model species: the promise of next generation sequencing for comparative immunology.

Next generation sequencing (NGS) allows for the rapid, comprehensive and cost effective analysis of entire genomes and transcriptomes. NGS provides approaches for immune response gene discovery, profiling gene expression over the course of parasitosis, studying mechanisms of diversification of immune receptors and investigating the role of epigenetic mechanisms in regulating immune gene expression and/or diversification. NGS will allow meaningful comparisons to be made between organisms from different taxa in an effort to understand the selection of diverse strategies for host defence under different environmental pathogen pressures. At the same time, it will reveal the shared and unique components of the immunological toolkit and basic functional aspects that are essential for immune defence throughout the living world. In this review, we argue that NGS will revolutionize our understanding of immune responses throughout the animal kingdom because the depth of information it provides will circumvent the need to concentrate on a few "model" species.

Differential effects of metal contamination on the transcript expression of immune- and stress-response genes in the Sydney Rock oyster, Saccostrea glomerata.

Environmental contamination by metals is a serious threat to the biological sustainability of coastal ecosystems. Our current understanding of the potential biological effects of metals in these ecosystems is limited. This study tested the transcriptional expression of immune- and stress-response genes in Sydney Rock oysters (Saccostrea glomerata). Oysters were exposed to four metals (cadmium, copper, lead and zinc) commonly associated with anthropogenic pollution in coastal waterways. Seven target genes (superoxide dismutase, ferritin, ficolin, defensin, HSP70, HSP90 and metallothionein) were selected. Quantitative (real-time) PCR analyses of the transcript expression of these genes showed that each of the different metals elicited unique transcriptional profiles. Significant changes in transcription were found for 18 of the 28 combinations tested (4 metals × 7 genes). Of these, 16 reflected down-regulation of gene transcription. HSP90 was the only gene significantly up-regulated by metal contamination (cadmium and zinc only), while defensin expression was significantly down-regulated by exposure to all four metals. This inhibition could have a significant negative effect on the oyster immune system, promoting susceptibility to opportunistic infections and disease.

Shotgun proteomics of coelomic fluid from the purple sea urchin, Strongylocentrotus purpuratus.

The purple sea urchin has a complex immune system that is likely mediated by gene expression in coelomocytes (blood cells). A broad array of potential immune receptors and immune response proteins has been deduced from their gene models. Here we use shotgun mass spectrometry to describe 307 proteins with possible immune function in sea urchins including proteins involved in the complement pathway and numerous SRCRs. The relative abundance of dual oxidase 1, ceruloplasmin, ferritin and transferrin suggests the production of reactive oxygen species in coelomocytes and the sequestration of iron. Proteins such as selectin, cadherin, talin, galectin, amassin and the Von Willebrand factor may be involved in generating a strong clotting reaction. Cell signaling proteins include a guanine nucleotide binding protein, the Rho GDP dissociation factor, calcium storage molecules and a variety of lipoproteins. However, based on this dataset, the expression of TLRs, NLRs and fibrinogen domain containing proteins in coelomic fluid and coelomocytes could not be verified.

Proteomic analysis of Sydney Rock oysters (Saccostrea glomerata) exposed to metal contamination in the field.

This study used proteomics to assess the impacts of metal contamination in the field on Sydney Rock oysters. Oysters were transplanted into Lake Macquarie, NSW, for two weeks in both 2009 and 2010. Two-dimensional electrophoresis identified changes in protein expression profiles of oyster haemolymph between control and metal contaminated sites. There were unique protein expression profiles for each field trial. Principal components analysis attributed these differences in oyster proteomes to the different combinations and concentrations of metals and other environmental variables present during the three field trials. Identification of differentially expressed proteins showed that proteins associated with cytoskeletal activity and stress responses were the most commonly affected biological functions in the Sydney Rock oyster. Overall, the data show that proteomics combined with multivariate analysis has the potential to link the effects of contaminants with biological consequences.

Time course proteomic profiling of cellular responses to immunological challenge in the sea urchin, Heliocidaris erythrogramma.

Genome sequences and high diversity cDNA arrays have provided a detailed molecular understanding of immune responses in a number of invertebrates, including sea urchins. However, complementary analyses have not been undertaken at the level of proteins. Here, we use shotgun proteomics to describe changes in the abundance of proteins from coelomocytes of sea urchins after immunological challenge and wounding. The relative abundance of 345 reproducibly identified proteins were measured 6, 24 and 48 h after injection. Significant changes in the relative abundance of 188 proteins were detected. These included pathogen-binding proteins, such as the complement component C3 and scavenger receptor cysteine rich proteins, as well as proteins responsible for cytoskeletal remodeling, endocytosis and intracellular signaling. An initial systemic reaction to wounding was followed by a more specific response to immunological challenge involving proteins such as apolipophorin, dual oxidase, fibrocystin L, aminopeptidase N and α-2-macroglobulin.

Proteomic discovery of biomarkers of metal contamination in Sydney Rock oysters (Saccostrea glomerata).

In the current study we examined the effects of metal contamination on the protein complement of Sydney Rock oysters. Saccostrea glomerata were exposed for 4 days to three environmentally relevant concentrations (100 µg/l, 50 µg/l and 5 µg/l) of cadmium, copper, lead and zinc. Protein abundances in oyster haemolymph from metal-exposed oysters were compared to those from non-exposed controls using two-dimensional electrophoresis to display differentially expressed proteins. Differentially expressed proteins were subsequently identified using tandem mass spectrometry (LC-MS/MS), to assign their putative biological functions. Unique sets of differentially expressed proteins were affected by each metal, in addition to proteins that were affected by more than one metal. The proteins identified included some that are commonly associated with environmental monitoring, such as HSP 70, and other novel proteins not previously considered as candidates for molecular biomonitoring. The most common biological functions of proteins were associated with stress response, cytoskeletal activity and protein synthesis.